Methods for labeling nucleotides, labeled nucleotides and useful intermediates

ABSTRACT

The present invention provides a labeled nucleotide having formula:                    
     where X is an aliphatic diamine, and the labeling moiety comprises a label and a spacer, where the spacer is coupled at one end to a platinum atom and at the other end to the label, which spacer comprises a chain having at least four atoms. The invention further provides a method for labeling a nucleotide, kits for labeling a nucleotide, and kits containing labeled nucleotides.

Continuing application: Divisional of Ser. No. 09/402,707, filed on Oct.8, 1999 now U.S. Pat. No. 6,338,943.

The invention relates to methods for labeling nucleotides using linkers(linking moieties between labels and bio-organic molecules, whichlinkers are based on platinum compounds).

Platinum (coordination) compounds have been considered interestingmolecules for a very long time. For a review of these compounds andtheir uses we refer to Reedijk et al. (Structure and Bonding 67,p.53-89, 1987). Especially Cis-platinum has received a lot of attentionas a possible anti-tumour drug. This anti-tumour reactivity of platinumcompounds originates from their having at least two reactive groups(preferably cis-oriented towards each other), which make it possible tocross-link DNA molecules, thereby inhibiting the replication of theseDNA molecules.

The British patent application 2 148 891 discloses cis-platinumcomplexes, which are six-coordinated. The platinum is attached to twohalogens or hydroxy groups, two additional halogens and to an ethylenediamine derived group, such as 1,2-diamino-2-methylpropane or1,2-diamino-2-methylbutane. The complexes are said to have an excellentanti-tumor effect.

In the European patent application four-coordinated complexes ofplatinum to 2,3-alkyl-1,4-butanediamine and two halogens are describedfor their anti-tumor effect.

Different four-coordinated platinum complexes are described in theEuropean patent application 0 386 243. The complexes comprise a diaminebidentate ligand and two 2-arylalkanoic acid or 3-aryl-2-oxoalkanoicacid ligands. These complexes are stated to have a strong growthinhibiting action on certain leukemia cells and are used for theironcostatic activity.

U.S. Pat. No. 4,207,416 discloses ethylenediamine-platinum(II)2,4-dioxopyrimidine complexes as having a high anti-tumor activity andlow mammalian toxicity.

A different use of platinum (coordination) compounds has been disclosedin PCT application (WO92/01699) wherein a platinum compound having onlytwo reactive moieties (denominated as leaving groups therein) is reactedwith a fluorescein to obtain a labeled platinum compound which can bind(non-covalently) to a nucleic acid, preferably at the N-7 position of aguanine residue.

Several methods for labeling nucleotides have been described in theliterature. For a long time, the standard method has been to useradioactive isotope labeling. However, there are a number of problemsassociated with the use of radioisotopes, such as potential healthhazards, disposal problems and instability problems.

In order to overcome these problems, Dale et al., Biochemistry, 14,(1975), 2447-2457, have proposed to use direct covalent mercuration as alabeling technique for nucleotides and polynucleotides. It was found,that cytosine and uracil may be mercurated at their C5-position undermild conditions. Further, Gebeyehu et al., Nucleic Acids Research, 15,(1987), 4513-4534, have reported that adenine and cytosine may belabeled with biotin derivatives through an aliphatic linker of from 3 to17 atoms.

A major drawback of these known methods is that they are not suitablefor labeling all different nucleotides. For instance, Dale et al.reported that their covalent mercuration method leads to negativeresults for adenine, thymine and guanine bases. In some cases, forexample when only a few residues of a certain nucleotide are present ina certain nucleic acid or when the terminating nucleotide residue of anucleic acid has to be labeled, it is desired to have at one's disposala method for labeling any nucleotide residue.

The present invention provides such a method. The method for labelingnucleotides of the invention comprises the steps of:

reacting a reactive moiety of a linker of the formula

wherein X represents any stabilizing bridge and wherein A and Brepresent the same or different reactive moieties, with an electrondonating moiety of a spacer, which spacer comprises a chain having atleast four atoms and at least one heteroatom in the chain, which spacerfurther comprises said electron donating moiety at one end of the chainand a reactive moiety at the other end of the chain; reacting thereactive moiety of said spacer with a label; reacting the other reactivemoiety of said linker with a nucleotide.

According to the invention, the linker may first be attached to thenucleotide and then to the spacer, or vice versa and the spacer mayfirst be coupled to the label and then to the linker or vice versa.

The reactive moiety of the spacer may be any reactive moiety that willenable the reaction between the spacer and the label in such a mannerthat a labeling moiety comprising a label and a spacer is formed, whichlabeling moiety is sufficiently stable.

The main purpose for labeling nucleotides is that these labelednucleotides can be incorporated in nucleic acid molecules. Modifiednucleotides, especially those wherein a (bulky) label is attached to thenucleotide, are often built-in into nucleic acids with a much lowerefficiency. The methods according to the invention result in labelednucleotides which are built-in into nucleic acids with a higherefficiency than the labeled nucleotides available to date. This isprobably for due to the selection of the spacers according to theinvention in combination with the platinum-based linkers according tothe invention.

The label to be used according to the invention is not critical. Inprinciple all labels which can be attached to a nucleotide and areemployed to date can be used. These labels may be radioactive labels,enzymes (which need reaction with a substrate to be detected), specificbinding pairs components such as avidin, streptavidin or biotin,biocytin, iminobiotin, colloidal dye substances, fluorochromes(rhodamin, etc.), reducing substances (eosin, erythrosin, etc.),(coloured) latex sols, digoxigenin, metals (ruthenium), metal sols orother particulate sols (selenium, carbon and the like), dansyl lysin,Infra Red Dyes, coumarines (amino methyl coumarine), antibodies, proteinA, protein G, etc. The invention has most benefits with bulkier labelssuch as biotin, avidin, streptavidin, digoxygenin or a functionalequivalent thereof.

The invention is not limited to nucleotides or nucleosides as such;derivatives and functional equivalents are also included. The usualnucleotides adenine, thymidine, cytosine, guanine and uridine arepreferred. Especially the purines are preferred which have a very goodincorporation rate.

For coupling of the spacer to the platinum linker an electron donatingmoiety is required. In a preferred method the electron donating moietyis an amine or a thiolate anion, which have both proven to be verysuccesful. It was found that aromatic amines, such as imidazoles orpurines, are capable of forming very strong bonds to platinum and thusare very suitable for use as the electron donating moiety.

The spacer is an important aspect of the present invention; it providesthe easiest coupling between label and linker. For avoiding sterichindrance in incorporation of the nucleotide into the nucleic acid itshould at least be four atoms long, preferably it is at least fourcarbon atoms long and has at least one heteroatom in that carbon chain.A heteroatom confers a certain amount of rigidity on the spacer. Thisrigidity provides an additional assurance that steric factors will notobstruct a convenient linking of a nucleotide and a label. It ispreferred that at least one heteroatom is an oxygen atom, whichpositively effects the hydrophilicity of the spacer.

Preferably, the spacer comprises no more than 20 carbon atoms in thechain, which is preferably an essentially non-branched chain, thuscausing no steric hindrance. The reason for this will be clear.

A highly preferred spacer is 1,8-diamino-3,6-dioxaoctane, hereinreferred to as Dadoo. Dadoo is a very flexible compound with a distalprimary amine group and a size that makes it very suitable for use asspacer according to the invention.

Another highly preferred spacer of the invention is an oligolysine or apolylysine. Due to their structure and conformation, these moleculescreate the most convenient environment for an optimal interaction amongthe actual label, the nucelotide and the platinum. An additionaladvantage of the use of lysine chains as the spacer is, that by alteringthe number of lysine units in the chain, the optimal conditions forspecific labels and nucleotides or nucleic acids can be attained. Givena certain application, the skilled person will easily determine how manylysine units are required for optimum results.

An especially interesting labeling moiety comprising a label and aspacer, has the formula

or the formula

wherein X represents any stabilizing bridge, Z and Z′ represent anon-leaving ligand and n is an integer of from 2 to 10.

Accordingly, the linker-spacer-label-system, or labeling substance, withthe labeling moiety of formula (II) or formula (III) has the formula

or the formula

wherein A, X, Z, Z′ and n have the above meanings.

The non-leaving ligands Z and/or Z′ are preferably an NH₃, NH₂R, NHR₂ orNR₃ group, wherein R represents an alkyl group having from 1 to 6 carbonatoms, because these ligands have an even smaller leaving-groupcharacter than other non-leaving ligands.

The interesting feature of using the labeling moieties having formulas(II) and (III) is that both the nucleotide and the actual label have thebenefit of being bonded directly to a platinum atom, while at the sametime these moieties are sufficiently far apart to avoid sterichindrance.

The linkers according to the invention preferably are platinum compoundswherein X represents an aliphatic diamine. In a preferred embodiment ofthe invention, one or both of the nitrogen atoms of the aliphaticdiamine are shielded. A suitable manner of shielding these nitrogenatoms consists of substitution with one or two alkyl groups of from 1 to6 carbon atoms, preferably methyl groups. This is advantageous in thathydrogen bonding between the triphosphate group of a nucleotide and thestabilizing bridge is prevented. Preferably, a diamine having 2-6 carbonatoms is use, most preferably an ethylene diamine group, which iswell-known for its stabilizing effect on this class of platinumcompounds. In this case, the linker has the formula

wherein G represents hydrogen or an alkyl group of from 1 to 6 carbonatoms and A and B represent the same or different reactive moieties.

The coupling or reactive moieties A and B are preferably the same andselected from the group consisting of NO₃ ⁻, SO₃ ⁻, Cl⁻, I⁻, or otherhalogens.

The invention of course also encompasses a labeled nucleotide obtainableby a method as disclosed above.

In addition, the invention encompasses a labeling substance for labelingnucleotides by a method as disclosed above. The labeling substance ofthe invention has the formula

wherein X and A have the above meanings and the labeling moietycomprises a label and a spacer as described above. Of course thelabeling substances of the invention can also be used for labelingpurposes other than labeling nucleotides. It was found that numerous(bio-) organic compounds, i.e. nearly every bio-organic molecule whichcontains an accessible sulphur or nitrogen atom, for example proteins,can be labeled with the platinum compounds of the invention.

A great advantage of the invention arises from the use of the platinumcompounds having formula (I) as linkers in the methods of preparinglabeled nucleotides according to the invention. These linkers can beprepared by very convenient and reliable methods.

In WO92/01699 the starting compounds disclosed for preparing labeledplatinum compounds are platinum(II)(ethylenediamine)dichloride andplatinum(II)(ethylenediamine)(Me₂SO)Cl. The first one can be obtainedcommercially, the second one (the preferred one) must be synthesized. Inthe dichloride compound, the Cl-ions are less readily substituted by alabel or a nucleotide, respectively. In the latter case, the totalnucleotide labeling time will be appreciably longer, up to severalhours, instead of several minutes.

The methods for preparing the linkers that are used in the method oflabeling nucleotides according to the invention are based on theselection of suitable starting compounds of the formula PtE₄ wherein Eis an electronegative group, preferably a halogen or NO₃ ⁻ or SO₃ ⁻. Thereaction, which is described in the examples, of these startingcompounds with e.g. ethylenediamine is very simple and efficient.Moreover, this reaction leads to very suitable symmetric intermediatecompounds for producing labeled nucleotides. A major advantage of usingthese compounds is that when a stabilizing bridge for the resultingplatinum compound has to be attached that no blocking reagents have tobe employed. Another advantage is that the resulting intermediatecompounds can again be labeled without the use of blocking agents.Therefore steps removing blocking agents can be eliminated completely.Furthermore the yields of these reactions are very high. Yet anotheradvantage of the use of these symmetrical starting compounds is that nomixtures of different resulting compounds can be formed, which mayinterfere with the following reaction and reduce yield or require extraseparation steps.

A very suitable intermediate compound according to the invention isplatinum(II)(ethylenediamine)(NO₃)₂. This substance can very easily beprovided with a suitable spacer and a labeling group, resulting inlabeling substances which can, through substitution of the remainingNO₃-group be linked to a nucleotide quite easily. Furthermore themethods for producing these compounds and the resulting compounds do notinvolve highly toxic substances such as DMSO.

The intermediate compounds can be labeled with any suitable label (alsoknown as marker) through a spacer as disclosed hereinabove.

Furthermore, the known advantages (from WO92/01699 for instance) are ofcourse also obtained with the present methods and compounds. Anotheradvantage of the platinum compounds is that they can be detected more orless directly by using the platinum as a nucleus for depositing silveror other metal crystals.

By binding the labeling substance to a nucleotide residue, DNA or RNAmolecules, be it single stranded or otherwise, can be easily detected,but it also allows for the production of probes for hybridizationtechniques wherein unlabeled DNA/RNA molecules hybridize to the labeledprobe. The platinum linker labeled nucleotides do hardly interfere withthe hybridization, if at all. Also, this technique obviates the use ofmodified nucleotides in preparing probes.

Nucleotides modified in accordance with the practices of this inventionand oligo- and polynucleotides into which the modified nucleotides havebeen incorporated or oligo- and polynucleotides that have been directlymodified using these novel platinum compounds may be used as probes inbiomedical research, clinical diagnostics and recombinant DNAtechnology.

Other advantages and embodiments of the invention will become clear fromthe following experimental part and the examples.

Experimental Synthesis of Intermediate Platinum Compounds

These compounds, i.e. the linkers having formula (I), may be prepared bya process which involves:

(a) reacting a compound having the structure:

with KI in a suitable solvent under suitable conditions so as to form aiodated platinum compound having the structure:

(b) reacting said iodated platinum compound with ethylenediamine in asuitable solvent so as to form a diethyleneamine iodated platinumcompound and represented by the formula Pt(en)I₂ and having thestructure:

(c) reacting said compound with AgNO₃, the reaction being carried out ina suitable solvent, under suitable conditions so as to form a compoundhaving the structure:

(d) reacting said compound with KCl in a suitable solvent under suitableconditions so as to form a compound having the structure:

(e) reacting said compound with AgNO₃ in a suitable solvent, undersuitable conditions so as to form a compound having the structure:

(f) recovering said compound as modified platinum starting compound forthe synthesis of hapten-bound Pt(en) compounds for use as DNA and/or RNAlabel.

EXAMPLE 1

A. Preparation of Pt(en)-diamine Starting Material

Preparation of Platinumethylenediamine(NO₃)₂: Starting Material.Pt(en)(NO₃)₂

All Reactions are Performed in the Dark

Dissolve 1 gram potassium tetrachloroplatinate (II), K₂PtCl₄ (2.4 mmol,Sigma) in 50 ml millipore (filtered water) and stir at room temperature.Add 10 equivalents of potassium iodide, KI (24 mmol, 3.999 g, Sigma).The colour of the solution will immediately turn from orange into darkred (K₂PtI₄), stir for 5 minutes.

Add one equivalent ethylenediamine (2.4 mmol, 160.8743 μl, Merck 11=0.9kg) after diluting 161 μl ethylenediamine in 5 ml millipore very slowlyto the platinum solution, mix this X solution for 1 hour at roomtemperature.

A yellow/brown precipitate, Pt(en)I₂, will be formed and the liquidstanding above should be clear.

Filter the solution through a 1.0 μm membrane filter(Schleicher&Schuell), wash the precipitate with millipore, ethanol anddiether (in this order). Dry the Pt(en)I₂ for at least 4 hours in avacuum dryoven at 37° C.

Weigh the dried Pt(en)I₂ (−1.07 g) and suspend it in 45 ml millipore/5ml aceton, the solution will be cloudy. Add 1.95 equivalent of AgNO₃(M=169.9, Sigma). Stir the reaction overnight at room temperature.

Filter the solution through a 1.0 μm membrane filter, the precipitate isSilveriodide, AgI, the filtrate should be clear.

Add to 0.5 ml of the filtrate, Pt(en)(NO₃)₂, an excess of KCl or NaCland make sure that no white precipitate is formed immediately afteradding the excess of KCl or NaCl. If no white precipitate (only a yellowone) is formed than add an excess of KCl or NaCl to the entire filtrate.After the yellow precipitate is formed filter the solution and wash theprecipitate (Pt(en)Cl₂) with millipore, ethanol and diether.

Dry the precipitate for at least 4 hours in a vacuum dryoven at 37° C.

Weigh the dry Pt(en)Cl₂ (M=326,1), and suspend it in 45 ml millipore/5ml aceton and stir the cloudy suspension. Add 1.95 equivalent AgNO₃ andstir the solution overnight at room temperature. The colour of thesolution will become white, due to the formation of AgCl.

Filter the solution in the dark and evaporate the filtrate to remove theaceton by rotation evaporation untill 25 ml of the filtrate is left. Thefiltrate is then freezedried. The product is checked by NMR or InfraredAbsorption Spectroscopy.

B. Preparation of Pt(tmen)-diamine Starting Material

Preparation of Platinum-N,N,N′N′-tetramethylethylenediamine(NO₃)₂:starting Material. Pt(tmen)(NO₃)₂

All Reactions are Performed in the Dark

Repeat the entire procedure of Example 1A, but useN,N,N′,N′-tetramethylethylenediamine instead of ethylenediamine.

EXAMPLE 2

A. Preparation of [Pt(en)(BioDadoo-NH₂)(NO₃)](NO₃)

Dissolve Pt(en)(NO₃)₂ (18.2 mg, 0.048 mmol) in 10 ml of Millipore waterand heat until dissolving. Dissolve BioDadoo (20 mg, 0.053 mmol,purchased from Boehringer Mannheim) in 5 ml of Millipore water. Add thetwo solutions together and adjust the pH to 8 by 0.1 N NaOH, react forat least 3 hours at 50° C. Isolate the end product by freeze drying.

B. Preparation of [Pt(tmen)(BioDadoo-NH₂)(NO₃)](NO₃)

Dissolve Pt(tmen)(NO₃)₂ (35 mg, 0.08 mmol) in 12.5 ml of Millipore waterand heat until dissolving. Dissolve BioDadoo (32 mg, 0.085 mmol) in 10ml of Millipore water. Add the two solutions together and adjust the pHby 0.1 N NaOH, react for at least 4 hours at 50° C. Isolate the endproduct by freeze drying.

C. Preparation of [Pt(en)(DigDadoo-NH₂)(NO₃)](NO₃)

Dissolve Pt(en)(NO₃)₂ (5 mg, 0.013 mmol) in 5 ml of Millipore water andheat until dissolving. Dissolve DigDadoo (9 mg, 0.016 mmol, purchasedfrom Boehringer Mannheim) in 5 ml of Millipore water. Add the twosolutions together and react for at least 4 hours at 50° C. Isolate theendproduct by freeze drying.

EXAMPLE 3

A. Preparation of a Labeled dGTP

Dissolve [Pt(en)(BioDadoo-NH₂)(NO₃)](NO₃) (9 mg, 0.012 mmol) in 2 ml ofMillipore water. Add 2′-deoxyguanosine-5′-triphosphate (2.3 mg, 0.004mmol) and adjust the pH to 6. Incubate for 24 hours at 50° C.,freeze-dry and redissolve in Millipore water (1 ml) and filter through amembrane filter. Apply the mixture to a FPLC with MonoQ and purify witha linear gradient from 100% Millipore water to 100% 1M NH₄HCO₃, collectand pool appropriate fraction and isolate by freeze drying. Dissolve theproduct in a 100 mM solution of triethylamine ammonium acetate (TEAA) (1ml) and apply to a Reversed Phase HPLC (C18) with a linear gradient from100% 100 mM TEAA to 50% 100 mM TEAA/50% 100 mM TEA/acetonitrile (l/lv/v), collect and pool the appropriate fraction and remove solvents byrepeated evaporation in vacuo Pass the product overa cation exchanger(Dowex) in the lithium form, isolate the product by freeze drying.

B. Preparation of a Labeled 5-AA-dUTP

Dissolve [Pt(en)(BioDadoo-NH₂)(NO₃)](NO₃) (6 mg, 0.008 mmol) in 2 ml ofMillipore water. Add 2′-deoxyuridine-5-aminoallyl-5′-triphosphate (2 mg,0.004 mmol) and adjust the pH to 8. Incubate for 24 hours at 50° C.,freeze-dry and redissolve in Millipore water (1 ml) and filter through amembrane filter. Apply the mixture to a FPLC with MonoQ and purify witha linear gradient from 100% Millipore water to 100% 1M NH₄HCO₃, collectand pool appropriate fraction and isolate by freeze drying. Dissolve theproduct in a 100 mM solution of triethylamine ammonium acetate (TEAA) (1ml) and apply to a Reversed Phase HPLC (C18) with a linear gradient from100% 100 EM TEAA to 50% 100 mM TEAA/50% 100 mM TEAA/acetonitrile (l/lv/v), collect and pool the appropriate fraction and remove solvents byrepeated evaporation in vacuo. Pass the product overa cation exchanger(Dowex) in the lithium form, isolate the product by freeze drying.

EXAMPLE 4

Reaction For Coupling Pt(en)-compounds to DNA

Typical Reaction For Labeling DNA Molecules with a Pt-Compound Accordingto the Invention.

5 μg of double stranded DNA is sonicated or DNase treated to yieldfragments of 300-500 bp. 6 μg of Pt(en)-compound is added and the volumeis adjusted to 50 μl with demineralised water. The reaction mixture isincubated at 65° C. for 1 hour. Non-bound Pt(en)-compound is blocked byadding 100 μl of a NADDTC solution. The Pt(en)-compound labeled DNA ispurified on a sphadex G-50 column. Readily labeled and purified DNA isstored at −20° C. or used directly in a DNA probe based assay.Pt(en)-compound labeled DNA probes can be stored at least 2 years at−20° C. without loss of activity and/or specificity. All applicationsmentioned are carried out with probes labeled according to thisprotocol.

EXAMPLE 5

Biotin Labeling of DNA Probes with [Pt(en)(BioDadoo-NH₂)(NO₃)](NO₃)(BioDadoo-ULS)

Introduction

The labeling method has been used to label DNA probes with Biotin for InSitu Hybridization (ISH). In this example labeling procedures includingthe protocols and data for quality control procedures are presented. ForBiotin labeling a plasmid cloned total DNA of Human Papilloma Virus type6 (HPV-6, 40% GC basepairs) was used.

Experimental Procedures

Plasmid DNA Preparation

Total DNA of Human Papilloma Virus type 6 was cloned into vector pSp-64.Plasmid DNA was transferred into E.coli (C-600) and plated ontoampicillin containing LB plates Single colonies were grown overnight inlarge culture. Plasmid DNA was isolated according to the method ofBirnboim and Doly¹, purified by Sepharose Cl-2B columnchromatography(Pharmacia) and checked for inserts by restriction-enzyme-analyses.Plasmid DNA concentration was determined by A260/280 absorbtion. Afterethanol precipitation the DNA was reconstituted in 10 mM TRIS/HCl pH7.2, 0.3 mM EDTA to a final concentration of 1 μg/μl (batch#150894).Subsequently this DNA was sonicated (Soniprep 150., MSE) for 3 times 10minutes (amplitude 5 microns) on ice.

The size of the resulting DNA fragments was determined by 2% agarose gelelectrophoresis and found to be in between 200-400 basepairs(batch#051094).

Plasmid DNA Labeling and Purification

Plasmid HPV-6 DNA was labeled with BioDadoo-ULS by mixing the followingreagents:

plasmid HPV-6 DNA (batch# 051094)  5 μl (1 μg/μl) BioDadoo-ULS labelingreagent  8 μl (1 μg/μl) (batch# BX940830) Demineralised water(<0.2/μS/cm) 37 μl

The 50 μl reaction mixture was incubated for 15 minutes at 85° C.

Excess of labeling reagent was captured by adding 50 μl sodiumdiethyldithiocarbamate (2% solution in demineralised water) andincubating for 30 minutes at room temperature. Unbound BioDadoo-ULS wasremoved, using a S300 HR microspin column (Pharmacia), by size exclusionchromatography. Eluent volume was adapted to 500 μl giving a 10 ng/μlbiotin HPV-6 probe concentration (batch#061094).

Quality Control for Detection Limits

The detection limit of the biotin probe of the invention was determinedby direct spot blot and reversed filterhybridization according to thefollowing protocols:

Direct Spot Blot

HPV-6 probe (batch#061094) labeled with biotin according to theinvention was 10-fold serially diluted into spot buffer comprising 900mM sodium chloride, 90 mM sodium citrate and 200 μg/ml single strandedsalmon sperm DNA giving a dilution series varying from 1000-0.1 pgbiotin probe per μl. 1 μl spots were applied onto nitrocellulosemembrane and incubated for 2 hours at 80° C. to bind the DNA. The biotinprobe was visualized using a streptavidin-alkaline phosphatase conjugate(Sigma) combined with. a NBT/BCIP precipitating substrate solution(Sigma) according to the following protocol:

Membranes were soaked in TBS solution containing 0.5% tween20 (TBST) for5 minutes.

Membranes were incubated with Strep-AP (3 DEA U/ml) in TBST for 15minutes at 37° C.

NC-membranes were washed 3 times 5 minutes in TBS solution followed by a5 minute wash step in demineralised water.

Membranes were incubated with NBT/BCIP substrate solution for 15 minutesat 37° C., subsequently washed in demineralised water and air dried.

Results

By using this method the detection limit of the biotin DNA probeaccording to the invention was found to be less than 1 pg.

Reversed Filterhybridization

HPV-6 DNA (batch#051094) was1 in 10 diluted in 0.1N NaOH, incubated at100° C. for 5 minutes and directly placed on ice for 5 minutes to makeDNA single stranded.

A 10-fold serial dilution was made in cold 0.1N NaOH to give a seriesvarying from 10,000-1 pg DNA per μl. 1 μl spots were applied onto Nylonmembrane (Boehringer Mannheim) and air dried.

HPV-6 DNA probe that was labeled with biotin according to the inventionwas diluted in 5× SSPE 0.5% SDS solution to yield a concentration of 200ng/ml.

This solution was incubated for 5 minutes at 100° C. and placed directlyon ice for 5 minutes.

Nylon membranes containing target DNA were soaked in 2× SSC for 5minutes and subsequently incubated with the single stranded probesolution for 2 hours at 37° C.

Excess of the biotin probe was removed by three chances in 2× SSPE 0.1%SDS for 10 minutes at 37° C. followed by a 5 minutes TBST incubation.

The biotin probe of the invention was visualized by performing the sameprotocol as described in the direct spot blot method.

Results

By using this procedure the detection limit of the biotin probeaccording to the invention was found to be less than 10 pg.

Performance in In situ Hybridization

The test material consisted of 6 μm paraffin sections of a HPV-6posotive cervical condyloma mounted on organosilane coated glass slides.

The following protocol was applied (unless otherwise stated steps are atroom temperature):

1 Paraffin sections were dewaxed in 3 changes of xylene and hydrated ingraded ethanol.

2 Sections were rinsed in TBST for 5 minutes.

3 Sections were digested in 0.25% pepsin in 0.1N HCl for 30 minutes at37° C., dehydrated in graded ethanol and air dried.

4 10 μl of probe solution was applied to a section and covered with acoverslip.

Probe solution consisted of biotin HPV-6 probe DNA labeled according tothe invention (batch#061094) in a concentration of 2 ng/μl dissolved inhybridization mixture comprising 0.6M NaCl, 0.06M sodium citrate, 35%formamid, 10% dextransulphate, 2,5× Denhardts and 10 μg/ml singlestranded salmon sperm DNA.

5 Slides were placed on a hot plate set at 95° C. for 5 minutes todenature probe and target DNA simultaneously.

6 Hybridization was performed by placing the slides in a humidifiedchamber at 37° C. for 2 hours.

7 Coverslips were removed and slides were washed in 3 changes of 15 mMNaCl, 1.5 mM sodium citrate and 5% formamid for 10 minutes at 37° C.

8 Slides were rinsed in TBST.

9 Sections were incubated with Streptavidin AP copnjugate (3DEAU/ml inTBST) for 15 minutes at 37° C.

10 Slides were washed in TBST (3×) and demineralised water (1×) for 5minutes.

11 Sections were incubated with NBT/BCIP substrated solution for 15minutes at 37° C.

12 Slides were washed in demineralised water (3×) for 1 minute andsections were mounted in glycerol/gelatin.

Results

By using the sections showed blue/purple precipitates at the sites ofHPV-6 infected cells and minor background in the remaining tissue.

Conclusions

The results demonstrate that DNA labeled according to the invention hasgood detection limits. The present method is very well suited forresearch, routine and for industrial production of labeled nucleicacids, as the method is fast and easy to perform, very sensitive, anddoes not include any enzymaic step, which makes it highly reproducibleand fitted for an overall low cost production. The method of theinvention offers a useful alternative equaling conventional non-isotopiclabeling methods.

General References

1. Maniatis T., Sambrook J., Fritsch E. F., Molecular Cloning, SecondEdition, Cold Spring Harbor Laboratory Press, ISBN 0-8769-309-6.

2. Keller G. H., Manak M. M., DNA probes, Stockton Press, ISBN0-333-47659-X.

Applications

1. The use of Pt-DNA linkers of the invention in the so called LIDIAtechnique: Linked DNA immuno Assay.

The LIDIA technique enables the quantitative analysis of small amountsof DNA (or RNA) e.g. after a PCR amplification of the starting material.The technique is sensitive and specific, due to the use of specificDNA(RNA) probes in accordance with the invention and easy to perform,because of the quick DNA(RNA) Pt-labeling steps of the invention.

Description of the Technique

The technique uses fast Pt labeling compounds of the invention to labelDNA(RNA) probes.

This technique is possible with 3 different approaches.

1. Linking DNA probes molecules to a surface by using a Pt compound inaccordance with the invention which cross-link DNA moleculesirreversibly to plastic, nylon or nitrocellulose. Detection of DNAtargets can then be accomplished by using classically labeled DNA/RNAprobes.(nick translation or chemical modification, random priming)

2. Linking a detectable group to the DNA, to render a DNA molecule intoa so-called DNA probe. Binding of DNA compounds to a surface can then beaccomplished by using classic techniques known to science (covalentlinking to specially treated microtiter plates, baking of DNA moleculesonto nitrocellulose or binding of DNA molecules to nylon membranes.

3. A combination of techniques 1 and 2

Approach 1

An immobilized DNA probe can be used to catch specific target moleculesin a sample by using a hybridization technique. Detection of formedhybrids can be done by using different techniques, e.g. a second labeledDNA probe can be used to hybridize with a different site on the targetDNA molecule to form a sandwich hybrid. The label can hen be detected byusing state of the art immunological detection and colouring techniques.

Approach 2

A volume containing (amplified) detectable DNA(RNA) is directly labeledaccording to the protocol in accordance with the invention.

Excess label is quenched by adding NaDDTC or Thioureum. This approachdistinguishes itself from other techniques by the fact that the targetmolecule is labeled in contrast to other assay were labeled DNA(RNA)probes are used to detect the target. The quick binding capacity of thePt-label compound of the invention enables a DNA binding step as aroutine step in a diagnostic test procedure (normal binding times are 60minutes at 65° C.).

A second step is performed in a microtiter plate precoated with a targetspecific probe. Incubation is allowed to the formation of stable“Labelled target” and probe hybrids. The direct labeling of targetmolecules enables the omission of laborious double hybridizationtechniques where one probe is used to catch the target and anotherlabeled probe is used to detect the immobilized target.

In this method the probes are covalently linked to the microtiter plateto the surface of the wells. The second incubation step has thecharacter of a liquid hybridisation and therefore can be performed veryrapidly. This is one of the main innovative features of this approach toquantitive DNA hybridisation techniques.

Approach 3

Both for the immobilization of DNA probes or DNA targets and for thelabeling of DNA probes and targets the newly developed Pt system can beused. These two DNA linking techniques can be combined into one assaywhere both the “catcher” and the “detector” are linked to a secondsubstance (either a detectable group like biotin, digoxigenin or acarrier surface like a plastic stick, microtiter plate or a membrane).

Examples of the Technique: the Detection of STD Related Microorganismsin Human Diagnostics (Chlamydia, Syfillis, AIDS, Herpes, Gonorrhoea,Hep. B,)

2. The Use of Pt-DNA Labels of the Invention in Combination with TestStrip Procedures and Formats. the “DNA Dipstick”.

The DNA dipstick technique enables the qualitative and semi-quantitativeanalysis of small amounts of DNA(or RNA) e.g. after a PCR amplificationor freely present in samples of body fluids (blood, urine, sweat etc.)

The technique is sensitive and specific, due to the use of specificDNA(RNA) probes and easy to perform because of the quick DNA(RNA)Pt-labeling steps according to the invention.

The universal labeling characteristics of the newly developed Pt labelcan be used in 3 ways to achieve a bound DNA(RNA) molecule.

1. It can be used to attach a detectable marker group to apolynucleotide sequence.

2. It can be used to attach polynucleotide sequences irreversibly to asolid phase (plastic, membranes, latex beads, hydrosols or microtiterplate wells).

3. A combination of 1 and 2

ad 1:

In this example there is a twofold approach to the detection of biolytesbiological analytes in test samples. Firstly a DNA probe can be labeledwith the newly developed Pt labeling compound. This labeled probe canthen be used to detect preformed hybrids on a membrane formed betweenthe target DNA sequence and a primary probe. It is essential in thismethod that the primary probe recognizes a different sequence on thetarget than the secondary Pt labeled probe. In practice, this can beachieved for instance with RNA hybridization were a POLY A probe is usedas a primary probe to immobilize all RNA (recognizable by its polyTtails) to a membrane.

The second approach differs slightly in that in this case the target canbe labeled in the test sample fluid, because of the fast and veryspecific Pt labeling characteristics. A procedure like this wouldcomprise a catch of the labeled target with an immobilized specificunlabeled DNA probe on a suitable membrane. Hence a dipstick version forDNA/RNA applications.

ad 2:

To immobilize DNA probes or target DNA, a non-labeled Pt compound (thatis a Pt compound with 2 free binding sites) can be used to act as abridge between DNA and the surface of carriers (plastic, membrane,microtiter plates etc.)

It greatly enhances the usability of DNA sequences as catcher moleculesin diagnostic assays, since there are little substances known to sciencethat bind readily DNA in a spontaneous way. Introducing this Pt bridgemolecule a wide field of new applications for the DNA technology hascome go within reach.

ad 3: a combination of example 1 and 2

General: the use of the Pt compound of the invention in latex orhydrosol assays is particularly interesting. The compound enables thecoupling of DNA molecules to small particles. The DNA molecules can behybridized to target material. A positive reaction is visualized by anagglutination of the particles, due to crosslinking of the DNA hybridparticle compounds.

A test like this can be made quantative, the rate of agglutination canbe tuned and measured at a specific wavelength. Especially goldparticles have the intrinsic characteristic that a shift in optimalwavelength occurs after agglutination.

3. Detection of Platinated DNA Probes of the Invention with the theSilver-Enhancement Technique

Platinated DNA/RNA probe can be employed in hybridisation methods todetect DNA/RNA sequences in sample material. The introduction of aplatinum compound at the site of the target enables the deposition of Agmolecules in a chemical reaction especially designed to reduce ionicsilver to metallic silver. At the site of a Pt nucleus a decompositionof metallic silver(black) occurs due to the catalytic effect of the Ptnucleus.

Ionic silver is reduced by a reducing agent (e.g. Na-borohydrid,Hydrochinon) in solution. In a constant ratio the amount of silverdeposited on the Pt is proportional to the length of the enhancementincubation. Visualisation of a non-visible Pt nucleus can beaccomplished by the empirical observation of the appearace of a blackspot in the test sample.

The black spots indicate the site of specific probes binding and thusthe site of specific target location. The technique enables a quick andeasy diagnostic procedure for the detection of various microorganismsand gene translocations/abnormalities.

What is claimed is:
 1. A labeled nucleotide having formula:

wherein: X represents an aliphatic diamine; and the labeling moiety comprises a label and a spacer, wherein the spacer is coupled at one end to the Pt atom and at the other end to the label, the spacer comprising a chain having at least four carbon atoms and at least one oxygen atom in the chain.
 2. The labeled nucleotide according to claim 1, wherein the aliphatic diamine has 2-6 carbon atoms.
 3. The labeled nucleotide according to claim 1, wherein the aliphatic diamine has the formula G₂NCH₂ CH₂ NG₂, wherein G represents H or an alkyl group of from 1 to 6 carbon atoms.
 4. The labeled nucleotide according to claim 1, wherein X represents ethylenediamine.
 5. The labeled nucleotide according to claim 1, wherein X represents N,N,N′,N′-tetramethylethylenediamine.
 6. The labeled nucleotide according to claims 1, wherein the spacer comprises no more than twenty carbon atoms.
 7. The labeled nucleotide according to claim 6, wherein the carbon atoms are non-branched.
 8. The labeled nucleotide according to claim 1, wherein the spacer is 1,8-diamino-3,6-dioxaoctane.
 9. The labeled nucleotide according to claim 1, wherein the label is radioactive.
 10. The labeled nucleotide according to claim 1, wherein the label is an enzyme.
 11. The labeled nucleotide according to claim 1, wherein the label is a component of a specific binding pair.
 12. The labeled nucleotide according to claim 11, wherein the specific binding pair is biotin and either avidin or streptavidin.
 13. The labeled nucleotide according to claim 1, wherein the label is a dye, a fluorochrome, or a reducing agent.
 14. The labeled nucleotide according to claim 1, wherein the label is digoxygenin.
 15. The labeled nucleotide according to claim 1, wherein the nucleotide is adenine, thymidine, cytosine, guanine, or uridine.
 16. The labeled nucleotide according to claim 1, wherein the nucleotide is adenine, thymidine, cytosine, and either guanine or uridine.
 17. The labeled nucleotide according to claim 1, wherein the nucleotide is a purine.
 18. A method for labeling a nucleotide comprising: providing a nucleotide; providing a labeling substance having formula VII,

wherein: X represents an aliphatic diamine; A represents a reactive moiety capable of reacting with the nucleotide, thereby attaching the nucleotide to the labeling substance when the reactive moiety reacts with the nucleotide; the labeling moiety comprises a spacer comprising an electron donating moiety bonded to the platinum atom, a chain having at least four carbon atoms and at least one oxygen atom in the chain, the chain attached to the electron donating moiety, and a label attached to the end of the chain distal to the electron donating moiety; and, reacting the reactive moiety with the nucleotide, thereby labeling the nucleotide.
 19. The method according to claim 18, wherein X represents an aliphatic diamine having 2-6 carbon atoms.
 20. The method according to claim 18, wherein X represents an aliphatic diamine having the formula G₂NCH₂ CH₂ NG₂, wherein G represents H or an alkyl group of from 1 to 6 carbon atoms.
 21. The method according to claim 18, wherein X represents ethylenediamine.
 22. The method according to claim 18, wherein X represents N,N,N′,N′-tetramethylethylenediamine.
 23. The method according to claim 18, wherein A represents NO₃ ⁻, SO₃ ⁻, Cl⁻, I⁻, other halogen or Me₂SO.
 24. The method according to claim 18, wherein A represents NO₃ ⁻.
 25. The method according to claim 18, wherein the spacer comprises no more than twenty carbon atoms.
 26. The method according to claim 25, wherein the carbon atoms are non-branched.
 27. The method according to claim 18, wherein the spacer is 1,8-diamino-3,6-dioxaoctane.
 28. The method according to claim 18, wherein the electron donating moiety is an amino group or a thiolate group.
 29. The method according to claim 28, wherein the amino group is an aromatic amino group.
 30. The method according to claim 28, wherein the amino group is an imidazole or purine group.
 31. The method according to claim 18, wherein the label is radioactive.
 32. The method according to claim 18, wherein the label is an enzyme.
 33. The method according to claim 18, wherein the label is a component of a specific binding pair.
 34. The method according to claim 33, wherein the specific binding pair is biotin and either avidin or streptavidin.
 35. The method according to claim 18, wherein the label is a dye, a fluorochrome, or a reducing agent.
 36. The method according to claim 18, wherein the label is digoxygenin.
 37. The method according to claim 18, wherein the nucleotide is adenine, thymidine, cytosine, guanine, or uridine.
 38. The method according to claim 18, wherein the nucleotide is adenine, thymidine, cytosine, and either guanine or uridine, or guanine and uridine.
 39. The method according to claim 18, wherein the nucleotide is a purine.
 40. A labeling substance having formula VII:

wherein: X represents an aliphatic diamine; A represents a reactive moiety; and the labeling moiety comprises a label and a spacer, wherein the spacer is coupled at one end to the Pt atom and at the other end to the label, the spacer comprising a chain having at least four carbon atoms and at least one oxygen atom in the chain.
 41. The labeling substance according to claim 40, wherein X represents an aliphatic diamine having 2-6 carbon atoms.
 42. The labeling substance according to claim 40, wherein X represents an aliphatic diamine having the formula G₂NCH₂ CH₂ _(NG) ₂, wherein G represents H or an alkyl group of from 1 to 6 carbon atoms.
 43. The labeling substance according to claim 40, wherein X represents ethylenediamine.
 44. The labeling substance according to claim 40, wherein X represents N,N,N′,N′-tetramethylethylenediamine.
 45. The labeling substance according to claim 40, wherein A represents NO₃ ⁻, SO₃ ⁻, Cl⁻, I⁻, other halogen or Me₂SO.
 46. The labeling substance according to claim 40, wherein the spacer comprises no more than twenty carbon atoms.
 47. The labeling substance according to claim 46, wherein the carbon atoms are non-branched.
 48. The labeling substance according to claim 40, wherein the spacer is 1,8-diamino-3,6-dioxaoctane.
 49. The labeling substance according to claim 40, wherein the electron donating moiety is an amino group or a thiolate group.
 50. The labeling substance according to claim 49, wherein the amino group is an aromatic amino group.
 51. The labeling substance according to claim 49, wherein the amino group is an imidazole or purine group.
 52. The labeling substance according to claim 40, wherein the spacer reactive moiety is NH₂.
 53. The labeling substance according to claim 40, wherein the label is radioactive.
 54. The labeling substance according to claim 40, wherein the label is an enzyme.
 55. The labeling substance according to claim 40, wherein the label is a component of a specific binding pair.
 56. The labeling substance according to claim 55, wherein the specific binding pair is biotin and either avidin or streptavidin.
 57. The labeling substance according to claim 40, wherein the label is a dye, a fluorochrome, or a reducing agent.
 58. The labeling substance according to claim 40, wherein the label is digoxygenin.
 59. The labeling substance according to claim 40, wherein the nucleotide is adenine, thymidine, cytosine, guanine, or uridine.
 60. The labeling substance according to claim 40, wherein the nucleotide is adenine, thymidine, cytosine, and either guanine or uridine.
 61. The labeling substance according to claim 40, wherein the nucleotide is a purine.
 62. A kit for labeling a nucleotide comprising: a nucleotide; and a labeling substance having formula VII,

wherein: X represents an aliphatic diamine; A represents a reactive moiety capable of reacting with the nucleotide, thereby attaching the nucleotide to the labeling substance when the reactive moiety reacts with the nucleotide; the labeling moiety comprises a spacer comprising an electron donating moiety bonded to the platinum atom, a chain having at least four carbon atoms and at least one oxygen atom in the chain, the chain attached to the electron donating moiety, and a label attached to the end of the chain distal to the electron donating moiety.
 63. The kit according to claim 62, wherein X represents an aliphatic diamine having 2-6 carbon atoms.
 64. The kit according to claim 62, wherein X represents an aliphatic diamine having the formula G₂NCH₂ CH₂ NG₂, wherein G represents H or an alkyl group of from 1 to 6 carbon atoms.
 65. The kit according to claim 62, wherein X represents ethylenediamine.
 66. The kit according to claim 62, wherein X represents N,N,N′,N′-tetramethylethylenediamine.
 67. The kit according to claim 62, wherein A represents NO₃ ⁻, SO₃ ⁻, Cl⁻, I⁻, other halogen or Me₂SO.
 68. The kit according to claim 62, wherein A represents NO₃ ⁻.
 69. The kit according to claim 62, wherein the spacer comprises no more than twenty carbon atoms.
 70. The kit according to claim 69, wherein the carbon atoms are non-branched.
 71. The kit according to claim 62, wherein the spacer is 1,8-diamino-3,6-dioxaoctane.
 72. The kit according to claim 62, wherein the electron donating moiety is an amino group or a thiolate group.
 73. The kit according to claim 72, wherein the amino group is an aromatic amino group.
 74. The kit according to claim 72, wherein the amino group is an imidazole or purine group.
 75. The kit according to claim 62, wherein the label is radioactive.
 76. The kit according to claim 62, wherein the label is an enzyme.
 77. The kit according to claim 62, wherein the label is a component of a specific binding pair.
 78. The kit according to claim 77, wherein the specific binding pair is biotin and either avidin or streptavidin.
 79. The kit according to claim 62, wherein the label is a dye, a fluorochrome, or a reducing agent.
 80. The kit according to claim 62, wherein the label is digoxygenin.
 81. The kit according to claim 62, wherein the nucleotide is adenine, thymidine, cytosine, guanine, or uridine.
 82. The kit according to claim 62, wherein the nucleotide is a mixture of adenine, thymidine, cytosine, and either guanine or uridine, or guanine and uridine.
 83. The kit according to claim 62, wherein the nucleotide is a purine.
 84. A kit for producing a labeled nucleic acid comprising: a labeled nucleotide having formula:

wherein X represents an aliphatic diamine; and the labeling moiety comprises a label and a spacer, wherein the spacer is coupled at one end to the Pt atom and at the other end to the label, the spacer comprising a chain having at least four carbon atoms and at least one oxygen atom in the chain; and unlabeled nucleotides.
 85. The kit according to claim 84, wherein X represents an aliphatic diamine having 2-6 carbon atoms.
 86. The kit according to claim 84, wherein X represents an aliphatic diamine having the formula G₂NCH₂CH₂NG₂, wherein G represents H or an alkyl group of from 1 to 6 carbon atoms.
 87. The kit according to claim 84, wherein X represents ethylenediamine.
 88. The kit according to claim 84, wherein X represents N,N,N′,N′-tetramethylethylenediamine.
 89. The kit according to claim 84, wherein the spacer comprises no more than twenty carbon atoms.
 90. The kit according to claim 89, wherein the carbon atoms are non-branched.
 91. The kit according to claim 84, wherein the spacer is 1,8-diamino-3,6-dioxaoctane.
 92. The kit according to claim 84, wherein the label is radioactive.
 93. The kit according to claim 84, wherein the label is an enzyme.
 94. The kit according to claim 84, wherein the label is a component of a specific binding pair.
 95. The kit according to claim 94, wherein the specific binding pair is biotin and either avidin or streptavidin.
 96. The kit according to claim 84, wherein the label is a dye, a fluorochrome, or a reducing agent.
 97. The kit according to claim 84, wherein the label is digoxygenin.
 98. The kit according to claim 84, wherein the labeled nucleotide is labeled adenine, thymidine, cytosine, guanine, uridine, or combinations thereof.
 99. The kit according to claim 84, wherein the unlabeled nucleotide is adenine, thymidine, cytosine, guanine, uridine, or combinations thereof.
 100. The kit according to claim 84, wherein the labeled nucleotide is a purine. 